The present invention relates to a method for determination of the gross nitrogen-mineralization rate of a soil sample. There is also provided a method for determining the amount of fertiliser to be applied to a soil and a test kit for readily determining a soil microbial enzyme activity present in a soil sample and correlating this activity with the corresponding gross nitrogen-mineralization rate of said sample.
More particularly, the invention provides for a novel and innovative method for determining the total release of plant available nitrogen (N) in soils. Soils contain nitrogen in both organic and inorganic forms. Both forms are potentially available for uptake by agricultural crops. However, organic forms must be converted to inorganic nitrogen before plant uptake.
The invention facilitates a determination of the rate at which organic nitrogen is converted to inorganic nitrogen or mineral nitrogen. This conversion is known as N-mineralization. The total release of plant available nitrogen in a soil, i.e. the gross N-mineralization rate quantifies the conversion in the soil of organic nitrogen to inorganic nitrogen.
The method of the invention can be used e.g. in a determination of the amount of a nitrogen-containing fertiliser to be applied to a soil such as e.g. a field of agricultural crops. Determination of the total release of plant available nitrogen in an agricultural soil is important when considering how much nitrogen-fertiliser to apply in order to obtain optimal growth conditions and at the same time minimising inorganic nitrogen-compounds such as nitrate from leaching out of the soil into ground or surface waters.
Nitrogen is present in the soil in both organic and inorganic forms. Nitrogen present in what is generally referred to as xe2x80x9csoil organic matterxe2x80x9d (SOM) such as macromolecular proteinaceous substances, humus, lignine, pectine, and the like, cannot readily be taken up be growing plants including agricultural crops. Nitrogen must be present in inorganic forms such as e.g. nitrate, ammonia in order to be taken up via the roots of the crops. The amount of inorganic nitrogen in the soil is generally much less than the requirement of the crops for nitrogen. It is therefore necessary to supplement nitrogen in the form of a suitable fertiliser.
An optimal fertiliser application shall facilitate optimal plant growth as well as minimal nitrogen leaching. Determination of the total release of plant available nitrogen in a soil is thus essential, when considering just how much nitrogen-fertiliser to apply, in order to achieve optimal growth conditions as well as preventing inorganic nitrogen-compounds such as nitrate from leaching out of the soil. Without any knowledge of the soils inherent capacity to release nitrogen to the crops, the farmer cannot accurately determine the amount of nitrogen-fertiliser to apply.
The conversion of organic nitrogen to nitrogen present in inorganic forms such as nitrate and ammonia is a dynamic and complex process, which cannot easily be accounted for. The process is influenced by e.g. climate, soil texture, total nitrogen, soil management, and the presence of soil microorganisms capable of degrading organic macromolecules and releasing e.g. nitrate and/or ammonia into the soil, and by the amount of nitrogen taken up by crops.
No simple method is available for measuring the gross N-mineralization rate. Thus, it has hitherto not been possible to account for nitrogen mineralization in fertiliser planning. Various methods for measuring directly or indirectly the amount and/or presence of organic and/or inorganic nitrogen-containing compounds in soil have been described in the prior art and briefly reviewed below.
Burton and McGill (1992) reported on changes in various components of a so called N-mineralization cascade. The changes studied included those of a specific component, such as a deaminase, as well as highly integrated components, such as a biomass. The selected soil was a Black Chernozemic seeded to barly (Hordeum vulgare L.) under field conditions. Changes in enzyme contents were related to soil ammonium in order to determine, if the microbial environment changed sufficiently to exert feedback control on N-mineralizing reactions, which would allow them to be detected. Histidase and protease were chosen as model systems for depolymerization and deamination, respectively, because information was already available on their control in pure culture studies, on histidine content and control of histidase in soil, and because assay procedures were readily available for soils. A correlation of an enzyme activity with gross N mineralization rates of a soil and problems associated with excessive use of fertilisers were not disclosed therein.
Burton and McGill (1989) characterised the stability of L-histidine NH3-lyase in soil by using a kinetic analysis and enzymatic assays in the presence of a biostatic agent. The objective was to employ a range of assay durations together with a kinetic analysis in order to examine the components of L-histidine NH3-lyase activity in soil, the stability of such components and the implications for the control of this enzyme. A correlation of an enzyme activity with gross N mineralization rates of a soil and problems associated with excessive use of fertilisers were not disclosed therein.
Galstyan and Vartanyan (Chem. Abstracts (1980), vol. 92, p. 494, 92:93207k) describe a method for determining an enzyme activity, i.e. arginase activity in soil samples by titration of ammonium ions. The results demonstrate a correlation of the content of humus with microbial activities, but do not evaluate or consider evaluating a correlation of an enzyme activity with gross N-mineralization rates of a soil.
SU-657344 relates e.g. to a calorimetric determination of inorganic nitrate and nitrate reducing enzymes in soil. The method is useful in evaluating nitrogen fixing soil bacterial activities. A correlation of an enzyme activity with gross N-mineralization rates of a soil and problems associated with excessive use of fertilisers were not disclosed.
SU-711470 relates to a determination of plant available nitrogen in a soil sample in order to evaluate a potential fertiliser requirement. A correlation of an enzyme activity with gross N-mineralization rates of a soil and problems associated with excessive use of fertilisers were not disclosed. Derwent Abstract WPI 96-391776 discloses a simple incubation method for determining the net mineralization potential of a soil. No enzymes are mentioned and consequently, a correlation of an enzyme activity with gross N-mineralization rates of a soil and problems associated with excessive use of fertilisers were not disclosed.
Derwent Abstract WPI 93-248897 discloses a method for calculating the net mineralization potential of a soil. A correlation of an enzyme activity with gross N-mineralization rates of a soil and problems associated with excessive use of fertilisers were not disclosed.
Cole (TIBTECH (1993), vol. 11, p. 368-372) describes the need to control environmental nitrogen in various ecosystems by means of exploiting microbial metabolisms, but no solution to the problem of how to reduce the excessive use of fertilisers is presented.
Benchemsi-Bekkari and Pizelle (Black locust nitrogen nutrition. Urea metabolism, relation with arginase and urease activities, Ph.D. thesis, (1993), Pascal no. 94-0041061) describe an acacia tree capable of assimilating nitrogen. Metabolism relating to the urea cycle is disclosed and a ratio of arginase activity to urease activity is determined. A correlation of arginase activity with gross N-mineralization rates of a soil and problems associated with excessive use of fertilisers are not disclosed.
Biosis Acc. No. 37066711 relates to a study of the effect of nitrogen-fertilisers on soil enzymes involved in nitrogen uptake and nitrogen metabolism in soil bacteria. A correlation of an enzyme activity with gross N-mineralization rates of a soil is not disclosed.
It is desirable to be able to determine in an accurate manner the significant variations in the release of inorganic nitrogen in a soil throughout the year. The essential question is how to accurately account for the gradual mineralization of organic nitrogen? Presently, no simple and inexpensive methods are available for measuring N-mineralization in soils, and only a limited knowledge of N-mineralization and crop nitrogen-demand has so far been extracted from field experiments analysing the response of crops to various nitrogen-fertilisers.
When calculating the amount of a fertiliser to apply to an agricultural field, the fanner must consider the nitrogen-demand of the crop in question. Crop nitrogen-demand has been determined for a wide range of different crops. As an example, a wheat crop usually requires ca. 180 kg nitrogen per hectare, while a spring barley crop usually requires ca. 120 kg nitrogen per hectare.
An illustration of the need to apply nitrogen-fertilisers to agricultural soils is given below. A 10 tonnes yield in winter wheat, which is often achieved by farmers in northern Europe, will have a content of some 200 kg nitrogen in the grains, a content of 40 kg nitrogen in the straws and a further content of 30 kg nitrogen in the roots and stubble. This gives a total nitrogen requirement of 270 kg nitrogen per hectare. The winter wheat may be grown on a soil containing as much as 5000 kg organic nitrogen per ha. 1-3% of this total amount of organic nitrogen, i.e. 50-150 kg nitrogen per hectare is mineralized each year, i.e. converted to inorganic nitrogen, which can be taken up by the plants. At the high level of 150 kg nitrogen mineralized per year, the crops will still require a supplement of nitrogen of about 180 kg nitrogen per hectare each year, assuming that 30-40% of such a supplement is leached or otherwise lost.
In another illustration of the significant contribution made by the N-mineralization process to the total amount of plant available nitrogen in the soil, a field of spring barley may need 120 kg nitrogen per year per ha. The process of N-mineralization may contribute as much as 50-100 kg, and consequently, the farmer, when taking this into account, will have to add the extra 20-70 kg by means of a fertiliser. The example illustrates two important things: Firstly, the N-mineralization is a major contributor of nitrogen compared to the amount of nitrogen provided in the form of a fertiliser, and secondly, it has not previously been possible to make an accurate assessment of the rate of N-mineralization.
Although the nitrogen-fertiliser demand of a certain crop on a particular field is primarily a function of the crop itself, the resulting fertiliser demand will obviously be affected by the fertility of the soil, i.e. the inherent capacity of the soil to release nitrogen to the crops in a readily assimilable form. The fertility of the soil thus depends on the rate of mineralization of organic nitrogen, that is the rate of gross N-mineralization. This is why it is important to provide an accurate estimate of this rate: It significantly influences the amount of fertiliser that needs to be applied to a field of crops. Furthermore, nitrogen-fertiliser demand and crop nitrogen-demand can be regarded as synonyms for the additional nitrogen fertiliser requirement of a certain crop on a particular field.
If insufficient amounts of minerals other than nitrogen were frequently found also to lead to growth limiting effects, the mass balance in the soil of such minerals would also be essential in fertiliser planning. However, nitrogen is very often the single most important growth limiting factor for most plants including agricultural crops. Accordingly, nitrogen must form an essential part of a fertiliser for crop production. It is a fact that the most important determinants of the amount of nitrogen-fertiliser required are the total amount of organic nitrogen present in a soil and the rate at which this organic nitrogen is converted to inorganic, i.e. plant assimilable nitrogen. Consequently, the rate of gross N-mineralization is not only the most important and significant parameter in fertiliser planning, it is also the most difficult process to include. Not surprising, N-mineralization is either not at all accounted for in conventional fertiliser planning, or alternatively, it is inaccurately accounted for due to a complete lack of methods providing a reliable and accurate determination of the rate of gross N-mineralization of a soil.
This lack of reliable and accurate methods poses a serious problem to the farmer: How to determine accurately the approximate amount of nitrogen-fertiliser to apply to a field of crops in order to facilitate an optimal crop growth and at the same time preventing e.g. nitrate leaching? The farmer may well acknowledge that a significant amount of nitrogen is already present in the soil, but how much nitrogen is present in a readily assimilable form. What is the rate of formation of inorganic nitrogen? Which legislative regulations impose restrictions on the use of nitrogen-fertilisers for the particular soil conditions and crops in question in order to prevent nitrogen leaching? The questions put to the farmer are manyxe2x80x94the answers surprisingly few and difficult to produce.
In summary, the fundamental information needed to facilitate an optimal fertiliser application is the knowledge of the amount of nitrogen required by the various crops on a particular field. The prime determinants of potential crop yield and thus crop nitrogen demand are the inherent crop properties, the nature of the soil and its inherent capacity to supply plant available nitrogen to the crops. This capacity is also termed the N-mineralization rate. N-mineralization in agricultural soils is therefore of fundamental agronomic importance. N-mineralization, i.e., the soils inherent capacity to supply nitrogen, is thus the prime determinant of additional fertiliser nitrogen demand (crop nitrogen demand).
It is a fact that nitrogen-fertilisersxe2x80x94if used excessivelyxe2x80x94will not only facilitate plant growth, but also lead to an undesirable build-up of nitrogen in the environment. Inorganic nitrogen-compounds such as nitrate, ammonia and nitrogen oxides may cause detrimental effects to both human health and sensitive, aquatic ecosystems. It is thus desirable to adjust the use of nitrogen-fertilisers to crop nitrogen demands and thus supplement a soil with an amount of nitrogen strictly required for crop growth. Also, from a production cost point of view, the purchase of nitrogen-fertilisers represent a major cost in contemporary agricultural productions, and the farmer has an economic incentive in trying to optimise the use of nitrogen-fertiliser. Further incentives for optimising fertiliser planning includes avoiding a possible tax on excessive use of fertilisers.
In order to reduce the amount of nitrogen leaching out into subsoil waters, mandatory crop rotation and fertiliser planning is required so as to avoid excess fertilisation and to further ensure, that the required minimum utilisation efficiencies of animal manures are achieved. To this purpose, farmers must each year submit a crop rotation plan, a fertiliser plan including an estimation of the need for nitrogen application according to economically optimal dosages, and specifications as to how the fertiliser requirement is being met by means of e.g. animal manure, other organic manures including waste products, or by the application of commercial fertilisers. The plans must also include a sketch map indicating the location and size in hectares of individual fields.
The total fertiliser application including the effective fraction of nitrogen contained in the animal manure must not exceed the crop demand set by the authorities. Moreover, the minimum utilisation efficiency of nitrogen in animal manure and other organic fertilisers established by authorities must be observed.
When the farmer prepares crop rotation and fertiliser plans, the nominal values for crop yield and nitrogen crop demand set by a number of crops by the authorities, must be applied. Nominal crop yields and thus nominal crop nitrogen demands for each crop in a variety of crop rotation plans may be introduced as a function of e.g. climate, soil type and access to irrigation. The nominal value of nitrogen application must also adhere to the limits established by an annual nitrogen prognosis. This prognosis is based on the so called xe2x80x9csquare grid netxe2x80x9d of representative sample points covering regions, where the soil mineral nitrogen content (inorganic nitrogen) is assessed. Typical corrections are in the order of plus or minus 5 kg nitrogen per hectare. In more extreme years the correction may average 10 to 15 kg nitrogen per hectare.
Legislation has been introduced in the European Union in order to reduce nitrate pollution. Directive 91/676/EEC, the so-called xe2x80x9cNitrate Directivexe2x80x9d, has the clearly stated objective to xe2x80x9creduce water pollution caused or induced by nitrates from agricultural sources and prevent further such pollutionxe2x80x9d.
Precision farming systems have been introduced in order to adjust the amount of a fertiliser to a specific soil condition and a specific crop demand in each subsection of a field, as compared to the conventional xe2x80x9cblanket dressingxe2x80x9d of fertilisers. The overall objectives of introducing such systems are partly aimed at reducing the amount fertiliser used, and thus improving the economics of the agricultural production, and partly aimed at achieving a more efficient application of the fertiliser in order to reduce the amount of nitrogen leaching.
Precision farming systems are presently based on indications of e.g. crop nitrogen-demand in subsections of an agricultural field. The subsections may have been introduced in order to facilitate the use of a so-called global positioning system (GPS). Accordingly, precision farming systems and the following adjustment of fertiliser application by means of a GPS are novel initiatives introduced by industry and farmers in order to try to optimise the use of nitrogen-fertilisers. However, the problem associated with the use of presently available precision farming systems is the lack of a simple and easy soil test to actually measure crop nitrogen demand. The direct result of using precision farming systems is the generation of a digital map of an agricultural field, which enables the farmer to use advanced fertiliser spreaders equipped with GPS and a personal computer (PC) in order to adjust fertiliser application in accordance with the nitrogen-content of a particular grid position and the specific crop nitrogen-demand for that grid position.
The N-mineralization process is mediated by a wide array of microorganisms present in the soil. Initially, such microorganisms will degrade nitrogen-containing polymers and macromolecules to nitrogen-containing oligomers and monomers. Some of these degradation products will be taken up and metabolised via, in principle, many different metabolic pathways present in different species of soil microorganisms. Further degradation products, such as nitrate, ammonia and urea, are generated by the soil microbial metabolism. Some of the products undergo further microbial and/or enzymatic processing. Examples are e.g. the uptake of released urea by some soil microorganisms (and the subsequent, intracellular metabolism of urea under the formation of e.g. ammonia), and the extracellular, enzymatic conversion of urea to carbon dioxide and ammonia, that takes place in the soil.
The microbially mediated process of N-mineralization is fundamentally a two step process. Firstly, the variety of organic nitrogen polymers present in soil is depolymerised by means of exoenzymes, i.e., enzymes produced by soil microorganisms and released into the soil. Such exoenzymes may e.g. hydrolyse large organic molecules to smaller molecules such as proteins to dipeptides or amino acids. Secondly, the smaller molecules are assimilated into microbial cells and further broken down by a set of microbial enzymes, which are internal to microbial cell walls. A detailed and conceptual model of N-mineralization is reviewed by Burton and McGill (1992). It is important to note that conventional wisdom dictates that the result of the N-mineralization is the direct formation of ammonia.
When urea is released into the soil solution it is rapidly converted to ammonia and CO2 if not re-immobilised into other microbia cells. Likewise, ammonia may also be immobilised into microbial cells. Both mineralization and immobilisation may thus take place at the same time but, importantly, the two processes do not take place in the same micro-pore space in the soilxe2x80x94the processes are essentially united in time but separate in space.
The total outcome of the two opposite processes, i.e., the net production and net consumption of soil inorganic nitrogen (including urea) is termed net N-mineralization. It may initially appear to be the net production of plant available nitrogen which is important for plant production, and simple concepts of soil fertility have been based on measurements of net N-mineralization by means of long-term incubation under laboratory conditions of soil samples in order to assess the potential net N-mineralization. However, soil fertility, and thus the potential for plant production, is more appropriately described by rates of gross N-mineralization.
Soils not recently being supplemented with organic materials and characterised by steady-state rates of gross N-mineralization and immobilisation, may be balanced so that e.g. 2.5 kg nitrogen per hectare per day is immobilised and 7.5 kg nitrogen per hectare per day is mineralized, generating a net rate of N-mineralization of 5 kg nitrogen per hectare per day. Although supplementing the soil with organic materials having a high carbon (C) to nitrogen (N) ratio, such as e.g. straws and stubbles plowed into the soil, no doubt stimulates N-immobilisation for some period of time, the process of N-immobilisation is confined to micro-pores with undecomposed plant material having a high CIN ratios. Gross N-mineralization, on the other hand, is widespread among micro-pores because soil organic matter (SOM) with a C/N ratio of ca. 10 is present in large amounts such as in the order of 5000 kg nitrogen per hectare.
Furthermore, the microbial cells produced as a result of the supplemented organic material will eventually die and contribute to the SOM substrate pool available for other microorganisms and, eventually, it will be mineralized. Accordingly, previously immobilised nitrogen-compounds will also be released into the soil and thus, in general terms, it may be stated that immobilisation of nitrogen in soil is confined in space and time, whereas the process of gross N-mineralization is a continuos process related to the total content and plant availability of SOM.
The sustained production in natural and managed ecosystems relies on replenishing the soil of nutrients removed herefrom by crop harvest, leaching, or by other processes. While natural ecosystems thrive because of well established nutrient cycling mechanisms, the cornerstone of stable agricultural ecosystem is replenishment of nutrients by either organic or inorganic fertilisers. The need to use fertilisers and the open type nutrient cycling in contemporary agricultural systems pose a particular challenge to the production of economically optimal crop yields while at the same time ensuring a minimal leaching to the surrounding environment.
The present invention relates to a method for determination of the gross N-mineralization rate of a first soil sample, said method comprising the steps of
i) determining the activity of a microbial enzyme of a functional ornithine acid cycle contained in said sample,
ii) determining
a) the activity of said enzyme in a second, predetermined soil sample, and
b) the corresponding gross N-mineralization rate of said second, predetermined soil sample, and
iii) determining the gross N-mineralization rate of said first soil sample on the basis of the gross N-mineralization rate corresponding to said activity determined in step ii).
Hereby, there is provided a quick and reliable method for measuring the gross N-mineralization rate of a soil sample. The method exploits that a microbial enzyme activity of a functional ornithine acid cycle present in said sample was surprisingly found to be directly correlatable with the rate of gross N-mineralization of that same sample.
Accordingly, the total amount of inorganic nitrogen available for plant uptake can be calculated based on a determination of the gross N-mineralization rate of a soil sample. The gross N-mineralization rate represents the overall conversion in the soil of nitrogen present in organic material to inorganic nitrogen such as e.g. nitrate and ammonia. Gross N-mineralization rates determined by using the method of the invention provides the farmer with a reliable estimate of the rate of inorganic nitrogen released into the soil, i.e. the amount of inorganic nitrogen measured e.g. in gram per kilogram soil per hour, or, in agronomic terms, e.g. in gram per hectare over a growing season.
In another aspect of the invention there is provided a method of determining the amount of fertiliser to apply to a soil. In yet another aspect there is provided a test-kit for determining the gross N-mineralization rate of a soil sample.
More specifically, the invention exploits the surprising discovery that the nitrogen of essentially all nitrogen-containing substances present in a soil, when being metabolised by soil microorganisms, is released via an arginine-urea metabolic pathway. The essence of the arginine-urea pathway is the enzymatic conversion of arginine to ornithine acid under the release of urea. The arginine-urea pathway forms part of a xe2x80x9cfunctional ornithine acid cyclexe2x80x9d, as the arginine-urea pathway leads to the regeneration of ornithine acid, which can then reenter the cycle and be converted to arginine.
Accordingly, any cyclical event involving a number of enzymatic steps leading to 1) the formation of arginine from ornithine acid and an ammonia donor and 2) the catalysis of arginine under the formation of urea and ornithine acid, may thus be termed a xe2x80x9cfunctional ornithine acid cyclexe2x80x9d. One example of a functional ornithine acid cycle is the conventional urea cycle cited in standard textbooks. However, variations of a conventional urea cycle may well exist among microbial soil organisms and such variations are intended to be comprised by the term xe2x80x9cfunctional ornithine acid cyclexe2x80x9d.
Consequently, the gross rate of N-mineralization can be measured by determining the xe2x80x9cflowxe2x80x9d of nitrogen through a functional ornithine acid cycle such as e.g. the conventional urea cycle. More particularly, the invention provides a quick, reliable and simple measure of said gross rate of N-mineralization by determining in a soil sample the content of an enzyme such as e.g. arginine deaminase (arginase), which is responsible for catalysing the deamination of arginine and the subsequent formation of urea and ornithine acid.
The method according to the invention can be used e.g. in determining the amount of nitrogen-containing fertiliser to be applied to a field of agricultural crops. This determination greatly facilitates the introduction of important new precision farming techniques because detailed mapping of N-mineralization rates, and thus crop nitrogen demands, within any subsection of any field, is now rendered possible. Although such precision farming technologies are already commercially available, a significant breakthrough of such systems awaits the development of a simple and inexpensive soil test such as the present N-mineralization test.
In summary, the gross N-mineralization rate facilitates a determination of the release of plant available nitrogen. This determination enables the farmer to include the release of nitrogen in fertiliser planning. Accordingly, the inclusion of a reliable N-mineralization rate facilitates the farmer in determining the amount of fertiliser to apply to a soil. If widely used among farmers, the present method will lead to a significant overall reduction in the amount of nitrogen-fertilisers being used and this will subsequently lead to a considerable reduction of e.g. nitrate leaching.